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The Study of Induction on Silenced Fragile X Mental Retardation-1 Gene Expression by Adenylat Cyclase Activator

Author: ZhangHongQin
Tutor: YaoYingMin
School: Southern Medical University,
Course: Pediatrics
Keywords: Fragile X , 1 mental retardation gene (FMR1) Dual luciferase reporter gene system Methylation cAMP activator
CLC: R749.94
Type: Master's thesis
Year: 2011
Downloads: 21
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Abstract

Background: Fragile X syndrome of fragile Ⅹ syndrome, Fra (X)] is the most common inherited mental retardation diseases. According to conservative estimates, there are at least 200,000 patients with fragile X mental retardation due to the patient's normal life, without the ability to live independently, and therefore cause a great burden on the family and the community. Fra (Ⅹ) pathogenic mechanisms are fragile x mental retardation One gene (gne of fragile the Ⅹ mental retardation-1 (CGG) n trinucleotide repeat sequence instability amplified its upstream promoter region of the FMR1) there CpG island aberrant methylation of these mutations in the FMR1 gene transcription was inhibited, reduced or absent lead to the encoding product fragile X mental retardation protein (fragile Ⅹ mental retardation protein, FMRP), the final performance Fra (X). Currently considered abnormal CpG island methylation is more important than the number of CGG repeats phenotype. Pietrobono R found in the case of DNA methylation, the amplification of the CGG repeat sequence itself does not prevent of FMR1 translation as well as of FMRP produce. Some reported cases, patients FMR1 gene (CGG) n has been amplified to the full mutation, but unmethylated CpG islands, FMRP still normal expression, so normal mental performance. Which prompted whether the methylation may be mainly due to the absence of the FMR1 gene transcriptional silencing and corresponding protein. FMRP reduced or absent, the final performance of the fragile X syndrome. FMRP is an RNA-binding protein, and shear on the transcriptional level by the transporters target RNA, RNA transporter, mRNA stability and translation level play an important regulatory role. Many studies have prompted FMRP affected by regulating the mRNA translation and protein synthesis to synapse formation and function. Regulation of protein synthesis, affect synapse development and brain plasticity in dendritic spines FMRP. Synapse, if FMRP lack, some of the proteins is excessive translation, lead to learning and memory-related long-term depression the enhanced (LTP). Thus the ultimate cause of mental retardation and behavioral problems. Another diagnosis were also detected the FMRP expression of rate than DNA testing to better reflect the level of patients with mental retardation. Short study of FMRP Fra (Ⅹ) pathogenesis, clinical diagnosis and treatment is essential. The Hwu research FMR1 gene promoter region there is a methylation-sensitive enhancer activity area (MSE) is closely related with the FMR1 gene transcription. The MSE and the cAMP response unit (CRE) base sequence overlapping CRE sequence ring adenosine monophosphate response element binding protein (CREB) binding sites. CREB is a nuclear transcription factor, with adjustment including memory function, including a wide range of biological functions, is necessary for long-term memory formation process of a ubiquitous regulatory molecules. Another study found that Fra (Ⅹ) closed the FMR1 gene of patients as well as in vitro model of intracellular cAMP water dropped by an average of the FMR1 gene (CGG) n trinucleotide fragment amplified by the size and content of cAMP was negatively correlated with the FMR1 protein-coding - Fragile X mental retardation protein increases intracellular cAMP production increased significantly. These data suggest that the FMR1 gene, there may be mutual regulation of the relationship between FMRP and cAMP. Our previous study two key enzymes of the cAMP metabolic processes in the FMR1 gene methylation closed: adenylyl cyclase (the adenylate cyclase AC) and phosphodiesterase (phosphodiesterase PDE) activity of found that intracellular cAMP levels in the FMR1 gene is closed, closed in the FMR1 gene group adenylate cyclase activity was significantly decreased, while no significant difference in the activity of phosphodiesterase. As a result, the proposed function of the FMR1 gene defects affect the adenylate cyclase activity, inhibition of adenylate cyclase activity may affect the main reason for the decrease in the the Fra (Ⅹ) patients intracellular cAMP level. This study attempts to methylation closed FMR1 gene cell models use drugs to improve the adenylate cyclase activity, improve the low levels of cAMP intracellular FMR1 gene, whether induced silencing the FMR1 gene transcription and protein expression? investigate the specificity of adenylate cyclase (AC) activator, based on - Frost succinylcholine (build FMR1 start forskolin, FSK) (?) the possibility of the show guide silence FMR1 gene transcription and protein expression and its effects; The mechanism of the promoter region of MSE / the CRE luciferase reporter gene system, further research AC agonist restart the FMR1 gene, as well as AC agonist activation the CRE sequence promoter methylation relations lay the foundation, and is expected to provide for the treatment of the disease new ideas and treatments. Methods: Cell culture: K-562 and Hela cells were with RPMI-1640 complete culture solution plus 10% newborn calf serum-free culture, the cells were subcultured temperature of 37 ° C and 5% CO2 saturated humidity incubator 2-3 days to replace the medium. Cell culture plus sodium nitroprusside were protected from light training. 2, making a closed cell model K-562 cells FMR1 gene: follows the principle of manufacturing model, namely: sodium nitroprusside (sodium nitroprusside, SNP) is a classic of nitric oxide (nitric oxide, NO) donor, generated through NO activation of DNA methyl transferase enzyme (DNA MeTase,) FMR1 promoter region CpG island MSE cytosine hypermethylation occurs, thereby blocking the combination of nucleic factor promoter suppressed FMR1 mRNA transcription. 2.1SNP concentration selected: the application of ordinary and Taqman fluorescence quantitative PCR observed FMR1 gene sealing effect, according to the literature is divided into the normal group, the SNP500umol / L group, SNP800umol / L group SNP1000umol / L group (corresponding to a final concentration of SNP were added) The cells were collected after 24h culture select the optimal concentration, according to the results. 2.2 to verify the effect of SNP closed FMR1 gene at the mRNA level: Ibid application of two PCR was used to observe the sealing effect of the FMR1 gene, located six time points {12h, 24h, 48h, 72h, 96h, 96h (72h replacement SNP after)} closed the last time, and the medium was changed after the observation disposable Join the SNP continued closure of the effect. 2.3 observe the SNP on the protein levels of FMRP expression: Application western blot method, were taken three time points (24h, 48h, 72h) observed FMRP expression. 3, observed the effect of the adenylate cyclase activator (FSK) on the the FMR1 closed mRNA levels restart: to adopt SNP1000umol / L closed gene, FSK (final concentration of 50μmol / L) was added after 24h culture, respectively 12h, 24h , 48h, 72h the cells were collected, and the normal control group, the application of the two PCR methods observed FMR1 gene closed after the restart effect. 4, the observation of the adenylate cyclase activator (FSK) restart FMR1 gene after closed FMRP expression: western blot method, in SNP1000umol, / L at culture after 24h FSK (final concentration of 50μmol / L) continued to train to 72h after the cells were collected, and the observation of FMRP expression. 5, luciferase vector construct overlapping fragments of 5.1. FMR1 gene promoter region MSE / CRE fishing take instructions conventional extraction of genomic DNA extraction kit: Press takala plasmid promoter fragment was amplified by PCR. T vector after PCR products connected to extract plasmid T-MSE, sequencing. 5.2 Construction of expression vector: In the T-MSE as the template, application of PCR method can be obtained with the restriction sites KPN I and Hind III the wild promoter fragment and plasmid was extracted, and then together with the PGL-4 vector plasmid with the restriction endonuclease Kpn Ⅰ and hind Ⅲ digested by PCR, restriction enzyme digestion and sequencing, to construct the expression vector. 6, the luciferase activity of the luciferase reporter gene system to detect 6.1 cells transfected: the logarithmic growth phase HeLa cells 24-well plate to a concentration of 105 per hole SHOP Lipofectamine2000 transfection reagent manual configuration transfection mixture (24 holes the board every hole amount): PGL-4/MSE promoter luciferase reporter plasmid or PGL-4 Basic 5ul Renilla plasmid 0.5ul lipo2000 3.5ul, and make co-transfected with further incubated. 6.2 Relative luciferase activity detection: collecting said cells described luciferase activity was determined with reference to the Dual-Luciferase Reporter Assay kit. Are under the same conditions of the above experiment was repeated 3 times, and the results were averaged. 7 Statistical analysis: all data for statistical analysis SPSS13.0 statistical software, poor median (MR) in addition to the the FMRP expression of the amount of data is not in line with the normal distribution, said outside, other data are expressed as mean standard deviation quantitative PCR results using 2 - △ △ CT value analysis, in addition to the SNP point-in-time data does not meet the homogeneity of variance between groups was used to compare the Kruskal-Wallistest completely randomized design multiple sample comparison, using the Bonferroni multiple comparisons (X SD); extrajudicial using completely randomized design comparing multiple samples One-Way ANOVA, multiple comparisons SLD law; analysis of Western blot data Bia-Rad software Density ratio, A value analysis does not match the normal distribution completely randomized design multiple sample comparison groups were compared using Kruskal-Wallis test for multiple comparisons using the Bonferroni method; relative luciferase activity detection data applications relative luciferase activity intensity value analysis, the groups were compared using two independent samples t inspection; above were taken P lt; 0.05 indicates difference was statistically significant. Results: 1, successfully produced closed cell model of the K-562 cells FMR1 gene 1.1 SNP in 500,800,1000 μmol / L concentration, there is a certain enclosed effect (F = 105.423, P lt; 0.001), the FMR1 gene of expression levels were down to pre-treatment 0.063,0.017,0.006 times, sealing effect is most obvious in the visible SNP1000μmol / L (P lt; 0.001), and once joined closure effect sustainable to 96H up to continue to foster change in 72h liquid Continuing closure effect. Closed FMR1 gene affect the the FMRP expression of protein levels 1.2 SNP: SNP closed In the FMR1 24h, 48h, after 72 h FMRP expression followed by decline, suggesting that the SNP closed FMR1 gene expression of the FMRP can greatly inhibited (H = 12.833, P = 0.012). 2, FSK FMR1 the closed gene mRNA level restart effect: FSK 24H when (F = 34.921, P lt; 0.001; P lt; 0.001) restart closed FMR1 gene and its expression level rose to a control group 0.133 times, and continued to 48H (F = 34.921, P lt; 0.001; P lt; 0.001), its expression is 0.138 times the control group. 3, FSK restart FMR1 closed gene of FMRP expression: FSK allows the FMRP expression was inhibited re-expression (H = 12.833, P = 0.012; P = 1.00) after 72 hours the FMRP expression of almost pretty with the control group . 4 luciferase vector construction: step sequence (MSE sequence the MSE sequence of restriction sites) sequencing preliminary identification, PCR, double digestion method and sequencing success, building a successful carrier. Luciferase reporter gene system fluorescent luciferase activity detection: PGL-4/MSE plasmid Renilla plasmid Total transfected hela cells measured the luciferase activity intensity value analysis, compared with the negative control group, the difference statistically significant, the plasmid was successfully transferred to the hela cells. Show that the overlapping sequence of MSE / CRE luciferase reporter gene was successfully constructed. Conclusions: 1. Successfully fabricated a closed-cell model of the K-562 cells FMR1 gene allows the closed after FMRl gene lowest expression levels fell to 0.006 times the pre-processing, FMRP expression levels fell to 0.477 times before treatment, to verify this The method of manufacturing the FRA (X) the feasibility of cell model. 2 confirmed FSK restart methylation closed FMR1 gene at the mRNA level, gene expression levels of up to 0.138 times the control group, and sustainable to 48 hours; accessible normal group found medication 72 hours after the FMRP protein expression level ; prompt specific adenylate cyclase activator can effectively induce methylation closed FMR1 gene transcription and protein expression, Fra (Ⅹ) treatment is expected to provide new ideas and therapeutic prospects. Successfully constructed overlapping sequence of MSE / CRE / of PGL-double fluorescent luciferase reporter gene system restart FMR1 gene mechanism for further research on AC agonist, and AC agonist activation relationship CRE sequence with promoter hypermethylation has laid foundation.

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